This invention relates to a process for determining the concentration of substances capable of promoting or inhibiting agglutination reactions and measuring the degree of reaction by quasi-elastic light scattering spectroscopy.
Agglutination reactions are widely used in biology and medicine to detect small quantities of antibody or antigen molecules. Agglutination reactions usually involve the in vitro aggregation of microscopic carrier particles which bear on their surface antigenic molecules. Aggregation occurs when antibody molecules specifically corresponding to the antigen are introduced into the solution of the carrier particles. The converse procedure of agglutinating antibody-coated particles with the appropriate polyhaptenic antigen molecules is also used. Some of the carrier particles which have been used are red blood cells, bacteria and polystyrene spheres. At low concentrations of the agglutination-inducing antibody or antigen (henceforth termed the agglutinator), small aggregates consisting of only a few carrier particles are formed. At higher concentrations of agglutinator the aggregates grow so large as to form visible clumps.
Conventionally, the appearance of this visible agglutinate has been taken as the criterion for the presence of the agglutinator. Clearly this detection criterion suffers from several defects. First, the formation of the grossly visible agglutinate requires a much larger concentration of agglutinator than needed to form small microscopic aggregates. Moreover, whereas the reversible formation of small aggregates is a specific and reproducible process, the appearance of macroscopic agglutinates is subject to many poorly controlled influences, such as the presence of foreign surfaces. In addition, the appearance of a grossly visible agglutinate is so qualitative a criterion that it is difficult experimentally to determine quantitatively the associated agglutinator concentration. Conventionally, the agglutinator concentration is determined by preparing a serial dilution of the agglutinator-containing solution. Then an aliquot of each dilution is mixed with a fixed amount of carrier particles (henceforth all the reagents, including carrier particles, used in fixed amount will be collectively termed the agglutinant) and the highest degree of dilution which still permits the formation of a visible agglutinate, is noted. This serves to indicate the concentration of agglutinator in the original solution. The agglutinator concentration can at best be determined to within a factor of two by this method.
Thus while the agglutination reaction, as conventionally performed serves as a specific and versatile means of detecting antigen or antibody molecules, it is severely limited in its application in that: (1) the process is not capable of providing an accurate quantitative measurement of either antigen or antibody concentrations and (2) the process may only be used for determining antibody or antigen concentrations which are sufficiently high so as to induce (or inhibit) macroscopically visible agglutination.
A present available alternative method for determining antigen concentration is the radioimmuno assay. In this method, a sample containing an unknown concentration of antigen is mixed with a fixed amount of antibody and a fixed amount of the antigen which has been radioactively labeled, usually with radioactive iodine. The resultant precipitate is recovered and its radioactivity is measured to determine the relative concentrations of the radioactive antigen and unknown antigen thereby to determine the concentration of the unknown antigen.
While this method is more sensitive than the method for visually determining agglutination reaction precipitate, it has many disadvantages. The radioimmuno assay requires the covalent linkage of a radio-isotope label to the species of antigen being measured. It is not always possible to attach this label to the desired class of molecules, and moreover the labelling process itself may distort the antigenic properties of the molecule so as to impair the sensitivity and specificity of the technique. In addition, the labelling process presents a radiation hazard and requires expensive shielding. Moreover, the labelled compounds are unstable and must be frequently prepared. Therefore, as a practical matter the use of radio-isotopes is so expensive, difficult and hazardous that in many applications the radioimmuno assay may not be used. In addition, the radioimmuno assay is not generally suitable for the measurement of antibody concentrations, and thus is undesirably limited to determining antigen concentrations.
In addition to determining antigen and antibody concentrations, the process of this invention can be used to determine the concentration of any substance capable of specifically promoting or inhibiting an agglutination reaction even where the formation of antigen-antibody bonds may not be involved in the agglutination process. For example, certain viruses are able to directly agglutinate red blood cells. Also, certain types of white blood cells of one individual may be able to agglutinate cells of another individual. The concentration of such viruses or white blood cells can be determined by effecting the appropriate agglutination reaction and determining the degree of reaction by quasi-elastic light scattering spectroscopy.